# US 2021/0275980 A1 · Composition and Methods for Producing Tobacco Plants and Products Having Reduced Tobacco-Specific Nitrosamines (TSNAs)

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- Number: US 2021/0275980 A1
- Document ID: US20210275980A1
- Status: Published application
- Kind: A1
- Application number: 16/838,762
- Filed: 2020-04-02
- Published: 2021-09-09
- Earliest claimed: 2016-10-07
- Assignees: Altria Client Services LLC
- Applicants: Altria Client Services LLC
- Inventors: Chengalrayan KUDITHIPUDI; Dongmei XU; James Strickland
- Web page: https://patentgrep.com/patent/US20210275980A1

## CPC

- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques}
- A01H 4/008: {Methods for regeneration to complete plants}
- A24B 13/00: Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff (reconstituted tobacco products A24B3/14)
- A24B 15/10: Chemical features of tobacco products or tobacco substitutes
- A24B 15/245: {Nitrosamines}
- B01F 33/45: Magnetic mixers; Mixers with magnetically driven stirrers
- B01F 33/452: using independent floating stirring elements
- B01F 33/50112: {of the syringe or cartridge type}
- B01F 35/754251: {reciprocating in the mixing receptacle}
- B01L 3/502: {with fluid transport, e.g. in multi-compartment structures}
- B01L 3/50825: {Closing or opening means, corks, bungs}
- C07K 14/415: from plants
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8225: {Leaf-specific, e.g. including petioles, stomata}
- C12N 15/825: {involving pigment biosynthesis}
- G01N 1/38: Diluting, dispersing or mixing samples
- H02N 11/006: {Motors}
- B01L 2200/0684: Venting, avoiding backpressure, avoid gas bubbles
- B01L 2300/043: Hinged closures
- B01L 2300/0672: Integrated piercing tool
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2400/0403: specific forces
- B01L 2400/043: magnetic forces
- B01L 2400/0478: pistons
- B01L 2400/0683: mechanically breaking a wall or membrane within a channel or chamber
- G01N 35/1079: {with means for piercing stoppers or septums}
- G01N 2035/00534: {Mixing by a special element, e.g. stirrer}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A24B: MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- H02N: ELECTRIC MACHINES NOT OTHERWISE PROVIDED FOR

## Abstract

The present disclosure provides approaches for reducing tobacco-specific nitrosamines (TSNAs) in tobacco. Some of these approaches include genetically engineering tobacco plants to increase one or more antioxidants, increase oxygen radicle absorbance capacity (ORAC), or reduce nitrite. Also provided are methods and compositions for producing modified tobacco plants and tobacco products therefrom comprising reduced TSNAs.

## Claims

### Claim at position 1

1-45. (canceled)

### Claim 46

Cured leaf of a modified tobacco plant, wherein said cured leaf comprises a reduced level of one or more tobacco-specific nitrosamines (TSNAs), an increased amount of protein activity in a protein comprising a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 13 and 23, and further comprises an increased level of one or more antioxidants, wherein said reduced and increased levels are compared to a control cured leaf of an unmodified tobacco plant of the same variety when grown and cured under comparable conditions; and wherein said increased amount of protein activity is a result of the modification in said modified tobacco plant.

### Claim 47 (depends on 46)

A tobacco product comprising the cured leaf of claim 46.

### Claim 48 (depends on 46)

The cured leaf of claim 46, wherein said cured leaf comprises an increased level of oxygen radical absorbance capacity (ORAC) compared to said control cured leaf.

### Claim 49 (depends on 46)

The cured leaf of claim 46, wherein said cured tobacco leaf comprises less than 2 ppm total TSNAs.

### Claim 50 (depends on 46)

The cured leaf of claim 46, wherein said cured tobacco leaf comprises less than 0.08 ppm 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), wherein the level of said total TSNAs is measured based on a freeze-dried cured leaf sample using liquid chromatograph with tandem mass spectrometry (LC/MS/MS).

### Claim 51 (depends on 46)

The cured leaf of claim 46, wherein said one or more antioxidants are selected from the group consisting of anthocyanidin, flavanone, flavanol, flavone, flavonol, isoflavone, hydroxybenzoic acid, hydroxycinnamic acid, ellagitannin, stibene, lignan, carotenoids, and glycyrrhzin.

### Claim 52 (depends on 46)

The cured leaf of claim 46, wherein said one or more tobacco-specific nitrosamines (TSNAs) are selected from the group consisting of N′-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine (NAT), and any combination thereof.

### Claim 53 (depends on 46)

The cured leaf of claim 46, wherein said modified tobacco plant is selected from the group consisting of a flue-cured variety, a Burley variety, a Maryland variety, a dark variety, and an Oriental variety.

### Claim 54 (depends on 46)

The cured leaf of claim 46, wherein said modification in said modified tobacco plant comprises a tobacco genome mutation or a transgene.

### Claim 55 (depends on 54)

The cured leaf of claim 54, wherein the level of said one or more TSNAs is reduced by at least 50% compared to cured tobacco leaf or a tobacco product from a control tobacco plant not comprising said transgene or said genetic modification.

### Claim 56 (depends on 54)

The cured leaf of claim 54, wherein said transgene encodes a protein comprising a sequence having at least 90% identity to SEQ ID NO. 13.

### Claim 57

Cured leaf of a modified tobacco plant, wherein said cured leaf comprises a reduced level of nitrite, an increased amount of protein activity in a protein comprising a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 13 and 23, comprises an increased level of one or more antioxidants, wherein said reduced and increased levels are compared to a control cured leaf of an unmodified tobacco plant of the same variety when grown and cured under comparable conditions; and wherein said increased amount of protein activity is a result of the modification in said modified tobacco plant.

### Claim 58 (depends on 57)

A tobacco product comprising the cured leaf of claim 57.

### Claim 59 (depends on 57)

The cured leaf of claim 57, wherein said cured leaf comprises an increased level of oxygen radical absorbance capacity (ORAC) compared to said control cured leaf.

### Claim 60 (depends on 57)

The cured leaf of claim 57, wherein said cured tobacco leaf comprises less than 2 ppm total TSNAs.

### Claim 61 (depends on 57)

The cured leaf of claim 57, wherein said cured tobacco leaf comprises less than 0.08 ppm 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), wherein the level of said total TSNAs is measured based on a freeze-dried cured leaf sample using liquid chromatograph with tandem mass spectrometry (LC/MS/MS).

### Claim 62 (depends on 57)

The cured leaf of claim 57, wherein said one or more tobacco-specific nitrosamines (TSNAs) are selected from the group consisting of N′-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine (NAT), and any combination thereof.

### Claim 63 (depends on 57)

The cured leaf of claim 57, wherein said modified tobacco plant is selected from the group consisting of a flue-cured variety, a Burley variety, a Maryland variety, a dark variety, and an Oriental variety.

### Claim 64 (depends on 57)

The cured leaf of claim 57, wherein said modification in said modified tobacco plant comprises a tobacco genome mutation or a transgene.

### Claim 65 (depends on 64)

The cured leaf of claim 64, wherein the level of nitrite is reduced by at least 50% compared to cured tobacco leaf or a tobacco product from a control tobacco plant not comprising said transgene or said genetic modification.

### Claim 66 (depends on 57)

The cured leaf of claim 57, wherein said transgene encodes a protein comprising a sequence having at least 90% identity to SEQ ID NO. 13.